Transforming growth factor alpha (TGFalpha), amphiregulin (AR) and cripto are proteins that are structurally and in some cases functionally related to epidermal growth factor (EGF) in that TGFalpha and AR can bind to the EGF receptor. TGFalpha has been implicated in the autocrine growth of a number of different human carcinoma cells such as breast and colon tumors. However, the regulation of expression of TGFalpha and interference with its biological activity have not been thoroughly examined; and relative levels of expression and biological function of AR and cripto in these malignancies are unknown. Present studies demonstrate that transformation of human mammary epithelial cells with a point-mutated c-Ha-ras protooncogene, but not with a c-erbB-2 oncogene, results in an increase in TGFalpha expression. Also, overexpression of human TGFalpha cDNA in these cells leads to in vitro transformation. Addition of an anti-EGF receptor blocking antibody or an anti-TGFalpha neutralizing antibody can partially or completely inhibit the growth of the ras or TGFalpha transformed mammary cells, suggesting the establishment of an external autocrine loop. Estrogens can increase TGFalpha mRNA and protein expression in estrogen-responsive human breast cancer cell lines like MCF-7 cells. Transient transfection assays in MCF-7 cells using a plasmid containing the TGFalpha promoter ligated to either the chloramphenicol acetyltransferase (CAT) or luciferase genes demonstrate that physiological concentrations of estrogens can induce a 10-to 100-fold increase in the activity of these reporter genes. This suggests that the TGFalpha promoter contains a cis-acting estrogen-responsive element (ERE) MCF-7 cells were infected with a recombinant amphotropic TGFalpha antisense expression vector. Expression of this antisense RNA leads to partial reduction in basal and estrogen-induced TGFalpha protein production and to an equivalent degree of inhibition of basal and estrogen-induced proliferation. Specific mRNA transcripts for AR and cripto were detected in approximately 70% of primary and metastatic colorectal tumors, but only 5% of normal colon or liver tissue expressed these genes. In contrast, cripto mRNA was not expressed in either normal or malignant human mammary tissue whereas AR mRNA was found in approximately 50% of these samples.